Open AccessResearch Establishment of an early liver fibrosis model by the hydrodynamics-based transfer of TGF-β1 gene Address: 1 Institute of Basic Medical Sciences College of Medicine,
Trang 1Open Access
Research
Establishment of an early liver fibrosis model by the
hydrodynamics-based transfer of TGF-β1 gene
Address: 1 Institute of Basic Medical Sciences College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, 2 Department of General Surgery, E-DA Hospital, I-Shou University, Kaohsiung 824, Taiwan, 3 Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan 717, Taiwan and 4 Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
Email: Kun-Lin Yang - s5889103@ccmail.ncku.edu.tw; Kuo-Chen Hung - hcc4723@yahoo.com.tw;
Wen-Teng Chang - wtchang@mail.hwai.edu.tw; Eric IC Li* - ericli@mail.ncku.edu.tw
* Corresponding author
Abstract
Background: Liver fibrosis represents a significant and severe health care problem and there are
no efficient drugs for therapy so far Preventing the progression of fibrogenesis and revival
endogenous repair activities is an important strategy for both current and future therapies Many
studies of liver fibrosis consist of animal testing with various hepatotoxins Although this method
is often used, the model at which cirrhosis or extensive fibrosis becomes irreversible has not been
well defined and is not representative of early-stage fibrogenesis We here report the establishment
of a transient and reversible liver fibrosis animal model which may better represent an early and
natural fibrotic event We used a high-speed intravenous injection of naked plasmid DNA of
transforming growth factor-β1 (TGF-β1) gene which is under the control of a
metallothionein-regulated gene in a pPK9A expression vector into the tail vein (the hydrodynamics-based transfer)
and fed the mouse with zinc sulfate (ZnSO4)-containing water simultaneously
Results: Using our hydrodynamics-based gene transfer model we found that upon induction by
ZnSO4, the serum TGF-β1 level in Balb/c mice and Sp1 transcription factor binding activity peaked
at 48 h and declined thereafter to a normal level on the 5th day In addition, mRNA and protein
levels of TGF-β1 in the liver were also upregulated at 48 h Furthermore, induction of TGF-β1
increased the α-smooth muscle actin (α-SMA), p-Smad2/3, hydroxyproline and collagen 1A2 (Col
1A2) levels in the liver, suggesting a significant liver fibrosis
Conclusion: Our results show that TGF-β1 in pPK9a-transferred mice liver with ZnSO4 feeding
can achieve a high expression level with significant fibrosis However, since TGF-β1 induction is
transient in our model, the fibrotic level does not reach a large scale (panlobular fibrosis) as seen
in the CCl4-treated liver Our model hence represents a dynamic and reversible liver fibrosis and
could be a useful tool for studying early molecular mechanism of fibrogenesis or screening of
antifibrotic drugs for clinical use
Published: 19 October 2007
Comparative Hepatology 2007, 6:9 doi:10.1186/1476-5926-6-9
Received: 22 May 2007 Accepted: 19 October 2007 This article is available from: http://www.comparative-hepatology.com/content/6/1/9
© 2007 Yang et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2The development of liver fibrosis, particularly in the
cir-rhosis stage, is associated with high morbidity and
mortal-ity rates [1] and at present the only curative treatment for
end stage liver cirrhosis is organ transplantation The
point at which cirrhosis or extensive fibrosis becomes
irre-versible has not been well defined [2], however, since liver
fibrosis is a continuous process in both gene expression
and histopathological alterations [3] Generally accepted
animal testing of liver fibrosis includes treatments with
hepatotoxins such as carbon tetrachloride (CCl4)
How-ever, after the cessation of the long-term treatment of CCl4
for more than 4 weeks, pathological changes in the liver,
such as inflammation, are reversed with the exception of
fibrosis [3] Many experimental long-term treatment
mod-els of liver fibrosis leading to cirrhosis have been useful
for testing drug effectiveness but further studies are
required to account for effects of disease treatment when
gene expressions, especially TGF-β1 gene, has not yet been
irreversibly altered [4]
TGF-β1, a 25-kD multifunctional cytokine, has been
dem-onstrated in a number of animal models to play a major
role in the pathogenesis and progression of fibrotic
dis-ease [5] Over expression of TGF-β1 presents not only an
early gene change in liver fibrosis but also a direct
connec-tion between oxidative stress and collagen upregulaconnec-tion in
the fibrosis event [6-8] Hepatic fibrosis results from a net
increased synthesis and decreased degradation of
extracel-lular matrix (ECM) proteins, whose most prevalent
pro-tein is Type 1 collagen (Col 1A2) TGF-β1 regulates ECM
accumulation in the liver via the generation of reactive
oxygen species (ROS) which stimulates calcium (Ca2+)
influx and induces the activation and contraction of
hepatic stellate cell (HSC) [8] The activated HSC in turn
secretes TGF-β1, further augmenting the autocrine
regulat-ing cycle
Another involved pathway is the activation of Smad
cas-cade The Col 1A2 gene expression is induced via the
phosphorylation of Smad2 and Smad3, a Smad
contain-ing complex is subsequently translocated into cell nucleus
[9] Studies have shown that synergistic cooperation
between Sp1 and Smad3/Smad4 is required for the
TGF-β1 response to the collagen gene expression and Sp1 is
found to play a critical role in the constitutive expression
of Col 1A2 [10] Cross-talk perhaps exists between these
two pathways [8]
The goal of our current investigation is to establish a liver
fibrosis model in which the spontaneous reversal of
fibro-sis is made possible at an early phase We used a
metal-lothionein-regulated TGF-β1 expression vector (pPK9a)
in which the fused TGF-β1 gene is under the control of an
inducible metallothionein promoter [11] This inducible
system has been reported to achieve a high-level of trans-gene expression in the liver when the system is accompa-nied with the concurrent presence of heavy metal [12] Hydrodynamics-based gene delivery has attracted a lot of attention in recent years [13] This procedure involves a large-volume and high-speed intravenous injection of naked plasmid DNA into the animal tail vein; the proce-dure represents an efficient, simple and convenient trans-fection method for laboratory animals The method especially allows the achievement of a high expression level of exogenous gene in liver [12-15] Combining a hydrodynamics-based gene delivery system and the met-allothionein-regulated pPK9a vector, we have established
a dynamic mouse liver fibrosis model In this model the level of TGF-β1 gene can be overexpressed with the pres-ence of zinc sulfate (ZnSO4) in the drinking water In induced state Col 1A2 and α-SMA, the two indicators of fibrosis and HSC activation, are also upregulated This model could be useful for studying the initial stages of liver fibrosis
Results and Discussion
Expression of TGF-β1 gene in hydrodynamics-based gene transferred mice
The level of TGF-β1 was assessed by using four independ-ent methods: analysis of TGF-β1 in plasma (Fig 1A), mRNA (Fig 1B) and protein (Fig 1C) in the liver and immunohistochemical staining in liver sections (Fig 1D) The results indicate that serum levels of TGF-β1 in pPK9a-transferred mice fed with ZnSO4 peaked at 48 h and were higher than the four control groups (Fig 1A) In the absence of ZnSO4 and pPK9a the serum TGF-β1 level is much lower than in their presence (Fig 1A) The serum TGF-β1 values fell between 600 and 900 pg/ml and were
5 to 15 times higher than the controls (Fig 1A); the peak was followed immediately by a decline at the 72nd h even when ZnSO4 was not withdrawn The level of TGF-β1 declined to its normal level on the 5th day and was no longer inducible on the 7th day (Fig 1E) The results are basically similar to that described by Herweijer et al who showed time course of gene expression after plasmid DNA gene transfer to the liver: expression of the transferred gene was very high on day1 after portal vein injection of plasmid but diminished quickly by day 2 and declined to low level after day 4 [16] Their induction was apparently transient as ours Moreover, Clouthier et al demonstrated that upon TGF-β1 gene transfer the mice showed similar pathological morphology in both liver and kidney [17]
As observed by them we also find high expression of TGF-β1 in the kidney (data not shown), although in this study
we just report our findings on fibrotic events in the liver Measurements of the other three analyses were taken at the 48th h and similar results were seen in mRNA and pro-tein expressions in liver tissue (Fig 1B, C and 1D)
Trang 3Prim-ers specific for porcine TGF-β1 in pPK9a were used for
distinguishing transcripts from endogenous mouse
TGF-β1 by RT-PCR assay (Fig 1B) TGF-TGF-β1 mRNA was detected
in mice with pPK9a-transfer (Fig 1B lane 1 and 2) but not
in those without (Fig 1B lane 3 and 4) However, in
west-ern blot a very trace amount of protein was detected in
mice without pPK9a-transfer (Fig 1C, lane 3 and 4) This
could be due to fact that the antibody used in the
experi-ment can recognize TGF-β1 from mice, human and
por-cine This trace amount of protein was barely detected in
immunohistochemical staining of the liver sections (Fig
1D II)
The high level of TGF-β1 was accompanied by a strong
activation of HSC, as indicated by high expressions of
α-SMA and p-Smad 2/3 (Fig 1C, lane 2) Prominent bands
of α-SMA and p-Smad 2/3 were also detected in
pPK9a-transferred mice that ingested water in the absence of ZnSO4 (Fig 1A, lane 3; 1B, lane 3; 1C, lane 1; and 1D I)
It was reported that a very low level of cadmium is con-sumed from the diet and this metal ion can induce endog-enous metallothionein to express to some extent [18] We suspect that the cadmium might cause the endogenous metallothionein induction as shown in Fig 1C (lane 1) However, the effect of ZnSO4 on pPK9a became more drastic as shown in Fig 1D where TGF-β1 was barely detected in D I (without ZnSO4) as compared to the bril-liant staining in D III (with ZnSO4) The levels of TGF-β1 expression in liver pPK9a-transferred mice treated with ZnSO4 were upregulated at day 2 and declined at day 3–5 (Fig 1E) Taken together, the results indicated that TGF-β1 can be markedly induced in pPK9a-tranferred mice treated with ZnSO4
Conditional regulation of TGF-β1 expression in mice
Figure 1
Conditional regulation of TGF-β1 expression in mice (A) Serum TGF-β1 levels in hydrodynamics-based gene
trans-ferred mice Values are represented as mean ± SD *, p < 0.01; compared with pPK9a alone (48 h); #, p < 0.05; compared with pPK9a + ZnSO4 (24 h); and, p < 0.05; compared with pPK9a + ZnSO4 (48 h); unpaired t-tests (B) RT-PCR analysis for TGF-β1
mRNA expression at 48th h in the liver of gene transferred mice after injection with pPK9a (C) Protein expression of TGF-β1 and α-SMA at 48th h in gene transferred mice after injection with pPK9a (D) Photomicrographs of immunohistochemical anal-ysis (upper panel) and bright field (lower panel) for TGF-β1 expression at 48 h in liver sections: I Ringer's solution + pPK9a and ZnSO4free II Ringer's solution only III Ringer's solution + pPK9a + ZnSO4 Bar = 0.2 mm (E) TGF-β1 protein expression between 1 to 5 days
Trang 4Histological and immunohistochemical analyses in
hydrodynamics-based pPK9a transferred mice
Liver sections were sampled from mice with pPK9a
trans-fer at the 24th and 48th h with water containing ZnSO4
(24, 48 h; Fig 2A and 2B) The liver samples looked paler
and stiffer than that of the control (Fig 2C and 2D) Using
Masson's trichrome staining, we noticed the liver sections
from mice with pPK9a transfer and ZnSO4 induction
exhibited a marked perisinusoidal deposition of ECM
found mostly in the direct vicinity of large vessels (Fig 3B,
I) and a distinct activation of HSC was also observed as
shown by α-SMA expression (Fig 3A, I) The distribution
and intensity of collagen and α-SMA were different from
that of the control livers where the two proteins were
barely detected (Fig 3) The sinusoids with an enlarged
diameter were observed in Fig 3B I–III, which was not
seen in the control (Fig 3B, IV) However, a bright red
patch is noticeable in TGF-β1-overexpressed liver (Fig
2B), which appears to be hemorrhagic (Fig 2B) The
"hemorrhage" was observed in most of our livers
overex-pressing TGF-β1 gene A similar situation of liver
hemor-rhage was observed by Clouthier et al who reported that
overexpression of TGF-β1 gene in mice caused not only
severe liver fibrosis but also in the extreme case
hemor-rhage and thrombosis [17] They attributed the extreme
situation to the results of overexpression of TGF-β1 and
Upregulation of α-SMA and ECM by gene transfer and ZnSO4 treatment in liver
Figure 3 Upregulation of α-SMA and ECM by gene transfer and ZnSO4 treatment in liver (A) Detection of α-SMA
by immunohistochemistry Forty eight h after hydrodynam-ics-based injection of pPK9a, the mice were sacrificed and the liver sections were subjected to immunostaining Dark brown granules represent α-SMA signals stained by α-SMA-specific antibody and indicated by arrows (B) Detection of ECM and collagen by Masson's trichrome staining The cyto-plasm was stained red and collagen fibers in ECM were blue-green The collagen signals were indicated by arrows Repre-sentative liver sections of α-SMA and collagen from experi-mental I-IV groups: (I) Ringer's solution + pPK9a + ZnSO4 48
h (II) Ringer's solution + ZnSO4 (III) Ringer's solution + pPK9a (IV) Mice without hydrodynamics-based injection Bar
= 0.2 mm
Observation of the liver
Figure 2
Observation of the liver Livers were obtained from mice
treated with Ringer's solution + pPK9a + ZnSO4 for 24 h (A),
Ringer's solution + pPK9a + ZnSO4 for 48 h (B), Ringer's
solution + pPK9a for 48 h (C), and vehicle (injection free) for
48 h (D)
Trang 5not the triggering cause of TGF-β1 overexpression We
agree with their proposal because we observed a quick
increased expression of TGF-β1 followed by a quick
decline Should the TGF-β1 expression was caused by liver
damage, we would not have found a prompt decline of
TGF-β1 (Fig 1A and 1E) More research is definitely
needed to clarify this controversy
Serum biochemical analysis
Forty eight hours after a hydrodynamics-based injection
of pPK9a, TGF-β1 was induced by ZnSO4 and triggered a
hepatic injury (Fig 2 and 3B), resulting in increased
alanine transaminase (ALT) levels in the serum of
approx-imately 6 times higher than that of the control groups
(Fig 4A)
Collagen expression in hydrodynamics-based fibrosis in
mice
The degree of fibrosis was assessed by using three
inde-pendent methods: the collagen quantitation by
measur-ing hydroxyproline content (Fig 4B), Col 1A2 mRNA
level in liver samples (Fig 4C) and the histopathological
analysis under light microscope (Fig 3B) The results
pro-vide factual epro-vidence that the pPK9a-transferred mice
upon induction by ZnSO4 could strongly elicit the
expres-sions of hydroxyproline and Col 1A2 over 3 folds and 200
folds, respectively, as compared to the normal control
groups (Fig 4B, C)
HSC activation plays a key role in liver fibrosis at the early
phase and activated HSC is accompanied with high
expressions of p-Smad2/3 and α-SMA proteins
[9,10,19,20] Our results mirror this fact as shown in Fig
1C where both proteins are markedly expressed as
com-pared with the controls Cirrhosis represents a later stage
of progressive scarring in chronic liver disease; it begins
with subendothelial or pericentral fibrosis (hepatic
fibro-sis) and progresses to panlobular fibrosis with nodule
for-mation (cirrhosis) [2] Our study demonstrates that liver
TGF-β1 of pPK9a-transferred mice with ZnSO4 feeding
can achieve a substantial increased expression level with
fibrosis However, since our TGF-β1 expression is
tran-sient, the fibrotic level does not reach a large scale
(pan-lobular fibrosis) as seen in the long term CCl4-treated liver
[Additional file 1] Although this CCl4-induced cirrhosis
model is commonly used, its effect is systemic and no
attempts are made to clarify the influence of CCl4 toxicity
[3] In this regard, our model is apparently different from
the CCl4 model with respect to TGF-β1; the life of TGF-β1
is transient, dynamic and overexpressed We also noticed
that the transient overexpression of TGF-β1 in the liver
leads to an increased deposition of ECM around the
ves-sels as well as along the sinusoids (Fig 3B) This finding is
consistent with the observation described by Ueberham et
al on transgenic animal models [21]
Observation of liver fibrosis in transgenic mice
Figure 4 Observation of liver fibrosis in transgenic mice (A)
Serum ALT levels (B) Hydroxyproline content in liver (C) Col 1A2 mRNA levels measured by real-time quantitative PCR All samples were collected 48 h after gene transfer and induction with ZnSO4 Values are represented as mean ± SD
*, p < 0.05; **, p < 0.001; compared with other groups;
unpaired t-tests.
Trang 6Gel electrophoretic mobility shift assays (EMSA) for Sp1
protein
To probe into the down steam effectors of TGF-β1 we refer
to the EMSA assay to see if Sp1 molecule specifically is
involved in the signaling pathway TGF-β1 being a strong
activator of ECM accumulation stimulates the Col 1A2
gene expression by inducing the binding of a Sp1- and
p-Smad2/3-Smad4-containing complex to Col 1A2
upstream promoter element (-330 bp to -286 bp and -271
bp to -255 bp; TGF-β1 responsive element; TbRE) which
contains a CAGA box Since Sp1 is a critical mediator of
Col 1A2 expression, we deem it prudent to examine if Sp1
was induced in pPK9a-transferred mice treated with
ZnSO4 and hence performed the supershift assay to
con-firm the Sp1 and Sp3 binding to TbRE Fig 5 shows that
the binding activity of Sp1 in liver increased at day 2 and
decreased at day 3–5 The pattern of Sp1 binding strongly
correlates with the expression levels of TGF-β1 (Fig 1E)
Since it has been reported that Sp1 is required for the early
response of Col 1A2 to TGF-β1 and maintenance of the
constitutive expression of Col 1A2 [20], our results
pro-vide direct epro-vidence confirming that our pattern of fibrosis
model is early, dynamic and reversible Moreover, Fig 3A
and 4C show that temporal activation of TGF-β1 and Sp1
is correlated with α-SMA and Col 1A2 expressions, a
find-ing consistent with previous reports [19,20]
Conclusion
The important role played by TGF-β1 in liver fibrosis has been well documented [5,7,8] and has been shown in transgenic mice model using pronuclear injection by standard technique [21] What we have shown here is a rapid fibrosis model with transient and reversible over expression of TGF-β1 and Sp1 transcription factor The expression is hence an early event We infer that the fibro-sis might also be transient and reversible However, the expression of transferred TGF-β1 appears to be systemic, not restricted to the liver as has been reported by our observation and by others But since hydrodynamic gene transfer coupled with metallothionein promoter and ZnSO4 induction has been reported to have higher expres-sion level of the exogenously delivered gene in the liver [12-15], our model is more unique to the liver and may have its usefulness for the clinical study of the prevention
of early stage of liver fibrosis
Methods
Animals
Mice of Balb/c strain were used All procedures of animal handling were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University Eight-week-old mice were used in all experiments and
were divided into five groups (n = 21), fed ad libitum
Enhancement of Sp1 binding activity by gene transfer accompanying with ZnSO4 treatment
Figure 5
Enhancement of Sp1 binding activity by gene transfer accompanying with ZnSO 4 treatment (A) Measurement of
Sp1 binding activity performed by EMSA Nuclear proteins were extracted from the livers of pPK9a-transferred mice adminis-trating ZnSO4-contained water for 1–5 days Two µg of nuclear extract was subjected to 32P-labelled probe and the Sp1-DNA complex was analyzed on a 4% native polyacrylamide gel (B) Confirmation of Sp1 and Sp3 binding by supershift assay Sp1 and Sp3 antibodies were added to the reaction mixtures for supershift assays The shifted and supershifted bands were indicated by arrows
Trang 7standard laboratory feed and water with or without 25
mM ZnSO4 plus 5% sucrose in the animal facility [22]
cDNA construction
TGF-β1 cDNA was constructed in pPK9a vector and was
under the regulation of metallothionein promoter Cys223
and Cys225 in the TGF-β1 pro-peptide were also converted
to serine, a mutation that results in dissociation of the
pro-peptide and secretion of bioactive TGF-β1 [11] It has
been found that this mutation does not alter TGF-β1
pro-duction but does yield a high proportion of mature 25
kDa dimer which is bioactive without acid activation
[11,22] pPK9a was a gift from Professor Paturu Kondaiah
of the Indian Institute of Science, India
Amplification and purification of plasmid DNA
The plasmid used for administration was purified with a
purification kit (Qiagen, Hilden, Germany) according to
the manufacturer's instructions
Hydrodynamics-based transfection
Ten µg of plasmid (pPK9a) were dissolved in 3.0 ml
Ringer's solution (NaCl 0.154 M, KCl 5.63 mM, and
CaCl2 2.25 mM) and injected into the mouse tail vein in a
short duration of 5–7 s according to the
hydrodynamics-based transfection protocol as described [14] ZnSO4 (25
mM) was dissolved in the drinking water to activate the
metallothionein promoter and stimulate TGF-β1
expres-sion [22,23]
Serum TGF-β1 levels
Blood was gathered by puncturing into retro-orbital veins
with a 27 gauge needle and reserved in a tube for 30 min
at 4°C Serum was separated by centrifugation at 2,640 g
for 3 min at 4°C TGF-β1 levels were determined by
enzyme-linked immunosorbent assays (ELISA) method
(DuoSet ELISA, R&D Systems, Minneapolis, MN)
Induction of TGF-β1 expression in liver
RNA was isolated from liver tissue by using Trizol Reagent
(GIBCO Life Technologies, Rockville, MD) TGF-β1
mRNA expression was detected by means of RT-PCR with
specific primers that distinguish porcine TGF-β1 transcript
from that of the mouse endogenous TGF-β1 PCR primers
specific for porcine TGF-β1 were:
5'-GAAAGCGGCAAC-CAAATC-3' and 5'-TGACATCAAAGGACAGCCAC-3'
Additional primers used for RT-PCR of
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene, were
5'-CCCT-TCATTGACCTCAACTAC-3' and
5'-CCACCTTCTTGATGT-CATCAT-3' All RT-PCR reactions were done for 35 cycles
[24]
Western blot analysis
For studying protein expressions of TGF-β1 and α-SMA, liver tissue was homogenized in a RIPA buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 1%
Nonidet P-40; 0.1% SDS; 1% Triton X-100 plus protease inhibitors Sigma, St Louis MO) and centrifuged as described [25]; supernatant was taken as a whole-cell lysate TGF-β1 and α-SMA were electrophoresed under non-reducing condition on a 12% SDS-polyacrylamide gel, transferred by electroblotting to a PVDF membrane, and visualized by immunostaining Anti-TGF-β1, anti-phospho-Smad 2/3 (Ser433/435-phosphorylated Smad2/ 3; p-Smad2/3), anti-Smad 2/3, anti-GAPDH and anti-α-SMA antibody (Santa Cruz Bio-technology, Inc., Santa Cruz, CA) were used as the primary antibodies Secondary antibodies were conjugated with horseradish peroxidase (Bio-Rad Laboratories) The signals were visualized by an enhanced chemiluminescence system (ECL, Amersham)
Hepatic hydroxyproline content
Hydroxyproline content was determined as reported with slight modification [25,26] Briefly, 100 mg of liver sam-ple were hydrolyzed in 6 M HCl at 110°C for 24 h After centrifugation at 2000 rpm at 48°C for 5 min, 2 ml of supernatant was mixed with 50 ml of 1% phenol-phthalein and 8 N KOH to pH7–8 A 5 ml sample was subjected to a spectrophotometer at 560 nm to determine the content of hydroxyproline
Biochemical analysis of plasma
Samples of 1 ml blood were gathered from the retro-orbital plexus of each mouse and immediately centrifuged
at 1,300 g at 4°C while plasma was kept at -20°C for liver
function tests ALT levels were measured using a colori-metric analyzer [27] (Dri-Chem 3000, Fuji Photo Film
Co, Tokyo, Japan)
Histological and immunohistochemical analysis of TGF-β1 and α-SMA expression
Mouse liver tissues were embedded in an optimal cutting temperature (OCT) compound (Miles Inc., Elkhart, IN) and frozen in liquid nitrogen Five µm-thick cryosections were made by using cryostats (Leica CM 1800, Nussloch, Germany) The sections were fixed with cold acetone and endogenous peroxidase was inhibited by 3% H2O2 in phosphate buffered saline (PBS) Then the sections were incubated with 5% blocking serum (normal serum of the species of the secondary antibody) For modeling the neg-ative control sections, the primary antibodies were substi-tuted for the appropriate classes and isotypes of normal immunoglobulins (Igs) Controls for nonspecific binding
of the secondary antibody were performed by replacing the solutions of the first step with PBS buffer TGF-β1 was revealed by using a rabbit polyclonal antibody (Santa
Trang 8Cruz Biotechnology, Inc., Santa Cruz, CA), and an
anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular
Probes, Eugene, OR) For the detection of α-SMA, a mouse
monoclonal antibody was used Signals were visualized
by anti-mouse IgG-HRP, horseradish peroxidase labeled
secondary antibody, and 3, 3'-diaminobenzidine
sub-strate (Vector Laboratories, Burlingame, CA) All sections
were viewed under a microscope (Leica Mikrosysteme
Ver-trieb GmbH, Bensheim, Germany) [21,25]
Masson's trichrome staining
Liver specimens were preserved in 4% paraformaldehyde
in phosphate-buffered saline and dehydrated in a graded
alcohol series Following xylene treatment, the specimens
were embedded in paraffin blocks and cut into 5 µm-thick
sections stained with Masson's trichrome as described
[21,25]
Quantitative real-time reverse transcription-polymerase
chain reaction (RT-PCR) analysis of collagen expression
Total RNA was isolated from the liver tissue by TRIZOL
reagent (Invitrogen, Carlsbad, CA, USA) RT was
per-formed as described [28] Quantitative real-time PCR was
performed with ABI Prism 7700 Sequence Detection
Sys-tem (Applied BiosysSys-tems, Foster City, CA) One µg of
cDNA was used in each PCR reaction The housekeeping
GAPDH was used as a reference gene for normalization,
and H2O was used as a negative control The primers for
the PCR reactions of Col 1A2 U08020 were:
5'-ACCTGT-GTGTTCCCTACTCA-3' and 5'-GACTGTTGCCTTCGCCTC
TG-3', the reaction was catalyzed by Taq polymerase
(Inv-itrogen Corp, Carlsbad, CA) SYBR Green I DNA-binding
dye generated the fluorescence signals during each of the
35 cycles, in proportion to the quantities of
double-stranded DNA (denaturation 15 s at 95°C, annealing 15 s
at 56°C and extension 40 s at 72°C) Each sample was
analyzed in triplicate Detection of the PCR products by
agarose gel electrophoresis confirmed the homogeneity of
the DNA products Relative quantitation was calculated
using the comparative threshold cycle (CT) method [as
described in the User Bulletin #2, ABI PRISM 7700
Sequence Detection System] Relative quantification of
the Col 1A2 transcript was compared to that of the
untreated negative control by the following formula:
and calculated ∆CT Col 1A2 = CT Col 1A2 - CT GAPDH
and ∆(∆CT) = ∆CT Col 1A2 - ∆CT negative control [29]
Preparation of nuclear extracts and EMSA
The preparation of liver nuclear extracts was based on the
method described by Chang and Huang with minor
mod-ifications [30] Fresh liver tissue of 0.1 g was homogenized
with a Polytron (Kinematica) in 1 ml of buffer A (10 mM HEPES (pH 7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM phenylmethylsulfonyl fluo-ride, 0.5 mM dithiothreitol, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 50 mM sodium fluoride, and 1 mM sodium orthovanadate), incubated on ice for 10 min and then gently shaken for 10 s The pellet of the crude nuclei was
collected by centrifugation at 12,000 g for 10 s,
resus-pended in 300 µl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM sodium chloride, 1.5 mM magne-sium chloride, 0.2 mM EDTA, 0.5 mM phenylmethylsul-fonyl fluoride, 0.5 mM dithiothreitol, 2 µg/ml leupeptin,
10 µg/ml aprotinin, 50 mM sodium fluoride, and 1 mM sodium orthovanadate) by vortex for 15 s, and then
incu-bated on ice for 20 min After centrifugation at 12,000 g
for 2 min, the supernatant containing the nuclear proteins was collected, quantified with BCA Protein Assay Reagent (Pierce), and stored at -70°C in aliquots For EMSA assay
we used the following oligonucleotides: consensus Sp1 (f) 5'-GTT GCG GGG CGG GGC CGA GTG-3' and consensus Sp1 (r) 3'-AAC GCC CCG CCC CGG CTC ACG-5' and labeled the probes with 32P-dCTP by fill-in method [30]
Statistics
Results were displayed by means of mean ± SD Statistical analysis was carried out by F-test (for confirming
homoge-neity of variances) and two-tailed Student's t-test (for eval-uating differences between means) P values lower than
0.05 (*) and 0.01 (**) were considered statistically signif-icant
Competing interests
The author(s) declare that they have no competing inter-ests
Authors' contributions
KLY performed most of the experiments and drafted the manuscript KCH participated in the design of the study WTC performed the EMSA and edited the manuscript EL coordinated the study and finally edited the manuscript All authors have read and approved the content of the manuscript
2−∆ ∆ ( CT)
Trang 9Additional material
Acknowledgements
We are greatly indebted to Dr Paturu Kondaiah of Indian Institute of
Sci-ence, India, for providing pPK9a and to Miss Renee Ting Yun Fang for her
editing assistance This work was partially supported by a grant from the
National Science Council, Taiwan (NSC 93-2622-B-006-002-CC3).
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Additional file 1
Liver sections of CCl 4 -induced fibrosis Comparative histology of liver
from mice treated with carbon tetrachloride (CCl 4 ) and
hydrodynam-ics-based transfer TGF-β gene Liver sections were stained with Masson's
trichrome (A) Mice were injected intraperitoneally with 0.3 ml CCl 4
solution (4% CCl 4 in corn oil) twice per week for 8 weeks (B) Ten µg of
plasmid (pPK9a) was dissolved in 3.0 ml Ringer's solution and injected
into the mouse tail vein in a short duration of 5–7 s The mice were fed
water containing 25 mM ZnSO 4ad libitum The collagen fibers peaked
at day 2 indicated by arrows Bar = 500 µm.
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